KR20210095413A - Medicinal composition for preventing or treating of disease induced by accumulation of beta amyloid - Google Patents

Abstract

The present invention relates to a composition which inhibits the aggregation of beta-amyloid by including a specific compound and inhibits toxicity by beta-amyloid by decomposing the aggregated beta-amyloid fibrils and oligomers, thereby fundamentally preventing and treating various diseases caused by the aggregation of beta-amyloid.

Description

Pharmaceutical composition for the prevention or treatment of diseases caused by aggregation of beta-amyloid

The present invention relates to a pharmaceutical composition for preventing or treating diseases caused by aggregation of beta-amyloid.

As life expectancy increases, the incidence of degenerative brain diseases is increasing significantly. In particular, senile dementia, including Alzheimer's disease, is recognized as a disease that increases the burden of not only the patient himself but also the people around him, as it is accompanied by functional impairment in which the overall cognitive and higher mental functions of language, learning, and intelligence are abnormally reduced. Although many target mechanisms have been elucidated, drugs used for dementia, including Alzheimer's disease, exist only with acetylcholinease inhibitors that can increase the amount of acetylcholine in the brain and drugs that are expected to slow the progression of the disease by blocking NMDA receptors. . In addition, the efficacy of these drugs is insignificant, and they are merely symptom-improving agents rather than therapeutic agents for diseases.

Korean Patent No. 2052097

An object of the present invention is to prevent aggregation of beta-amyloid and to provide a composition capable of fundamentally preventing and treating diseases caused by aggregation of beta-amyloid by decomposing the aggregate.

1. A pharmaceutical composition for preventing or treating a disease induced by aggregation of beta-amyloid, comprising a compound of Formula 1 below:

[Formula 1]

(Wherein, R ₁ , R ₂ , and R ₃ are each independently hydrogen, a hydroxyl group, an alkoxy group having 1 to 3 carbon atoms, or glucopyranosyl; at least one of the three is not a hydrogen atom or a methoxy group ).

2. The composition of the above 1, wherein the compound of Formula 1 is at least one compound of Formulas 2 to 5 below:

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

3. The composition of the above 1, wherein the compound of Formula 1 is a compound of Formula 3 or 4 below:

[Formula 3]

[Formula 4]

.

.

4. The composition of 1 above, wherein the disease is Alzheimer's dementia, Down's syndrome, amyloid angiopathy, systemic amyloid disease, Dutch type amyloidosis or inclusion body stomatitis.

5. Health functional food for preventing or improving diseases caused by aggregation of beta-amyloid in the brain, comprising the compound of Formula 1 below:

[Formula 1]

(Wherein, R ₁ , R ₂ , and R ₃ are each independently hydrogen, a hydroxyl group, an alkoxy group having 1 to 3 carbon atoms, or glucopyranosyl; at least one of the three is not a hydrogen atom or a methoxy group ).

The composition of the present invention can inhibit the aggregation of beta-amyloid and inhibit the toxicity caused by beta-amyloid by decomposing the aggregated beta-amyloid fibrils and oligomers. Therefore, it is possible to fundamentally prevent and treat various diseases caused by aggregation of beta-amyloid.

1 shows the inhibitory effect of beta-amyloid aggregation of Biflorin and its derivatives.
Figure 2 shows the decomposition effect of the beta-amyloid aggregates of Biflorin and its derivatives.
Figure 3 shows the inhibitory effect on the formation of beta-amyloid oligomers of Biflorin derivatives.
Figure 4 shows the cytotoxicity inhibitory effect of beta-amyloid of Biflorin derivatives.

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition for preventing or treating diseases caused by aggregation of beta-amyloid.

The composition of the present invention includes a compound of Formula 1 below.

[Formula 1]

(Wherein, R ₁ , R ₂ , and R ₃ are each independently hydrogen, a hydroxyl group, an alkoxy group having 1 to 3 carbon atoms, or glucopyranosyl; at least one of the three is not a hydrogen atom or a methoxy group ).

The present inventors confirmed that the compound of Formula 1 inhibits the aggregation of beta-amyloid and decomposes the aggregate, whereby the compound may exhibit preventive or therapeutic efficacy against various diseases caused by the aggregation of beta-amyloid.

The compound of Formula 1 may be specifically a compound of Formulas 2 to 5, and more specifically, a compound of Formula 3 or 4.

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

The disease to be prevented or treated by the composition of the present invention may be any disease that can be caused by aggregation of beta-amyloid without limitation, for example, Alzheimer's dementia, Down's syndrome, amyloid angiopathy, systemic amyloid disease, It may be Dutch type amyloidosis, inclusion body stomatitis, or the like.

The pharmaceutical composition of the present invention may be provided as a pharmaceutical composition including an active ingredient alone or one or more pharmaceutically acceptable carriers, excipients or diluents.

As used herein, the term “pharmaceutically acceptable” refers to exhibiting non-toxic properties to cells or humans exposed to the composition.

In the present invention, the administration route of the pharmaceutical composition is not limited thereto, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical , sublingual or rectal.

The composition of the present invention can be administered orally or parenterally, and when administered parenterally, it is preferable to select an injection method for external application or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. , but is not limited thereto.

The preferred dosage of the composition of the present invention varies depending on the condition and weight of the patient, the severity of the disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the composition is preferably administered at 0.01 to 1000 mg/kg/day, preferably at 0.1 to 500 mg/kg/day, but is not limited thereto. The administration may be administered once a day, or divided into several administrations. The above dosage does not limit the scope of the present invention in any way.

In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose. Or it is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycero geratin, and the like can be used.

In addition, the present invention relates to a health functional food for preventing or improving diseases caused by aggregation of beta-amyloid.

The composition of the present invention includes a compound of Formula 1 below.

[Formula 1]

(Wherein, R ₁ , R ₂ , and R ₃ are each independently hydrogen, a hydroxyl group, an alkoxy group having 1 to 3 carbon atoms, or glucopyranosyl; at least one of the three is not a hydrogen atom or a methoxy group ).

The compound of Formula 1 may be specifically a compound of Formulas 2 to 5, and more specifically, a compound of Formula 3 or 4.

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

The disease to be prevented or improved by the health functional food of the present invention may be any disease that can be caused by aggregation of beta-amyloid without limitation, for example, Alzheimer's dementia, Down's syndrome, amyloid angiopathy, systemic amyloid disease, Dutch type amyloidosis, inclusion body stomatitis, and the like.

The health functional food of the present invention may be formulated as one selected from the group consisting of tablets, pills, powders, granules, powders, capsules and liquid formulations, further comprising one or more of carriers, diluents, excipients and additives. Foods to which the extract of the present invention can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods.

Additives that may be further included in the present invention include natural carbohydrates, flavoring agents, nutrients, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), coloring agents, fillers (cheese, chocolate, etc.), At least one component selected from the group consisting of facic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonation agents, and pulp can be used. .

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the flavoring agent, natural flavoring agents (taumatine, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the composition according to the present invention may contain the pulp for the production of natural fruit juices and vegetable beverages. These components may be used independently or in combination.

Specific examples of the carrier, excipient, diluent and additive include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, stearic acid At least one selected from the group consisting of magnesium and mineral oil is preferably used.

When formulating the health functional food of the present invention, it is prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant that are usually used.

Hereinafter, examples will be given to describe the present invention in detail.

Example

1. Experimental method

1) Drugs and reagents

Beta-amyloid 1-42 was purchased from Anaspec. Biflorin derivatives were synthesized and provided by the inventor, Professor Namjoong Kim. As for the other reagents, the highest grade commercially available was used.

2) Beta-amyloid aggregation inhibition and aggregate decomposition effect

A method for confirming the degree of protein aggregation in vitro was used. Thioflavin T (ThT), a benzothiazole dye, was purchased from Sigma (T3516) and dissolved in DPBS. Amyloid β 1-42 (Aβ42) protein was dissolved in DMSO at 1 mM and diluted with DPBS to 100 μM. Biflorin and its derivatives were dissolved in DMSO and used. In order to check the degree of Aβ42 aggregation, the amount of Aβ42, biflorin and derivatives were mixed 1:1 and put in a black Eppendorf tube and incubated at 37 °C for 24 hours. The final concentrations were Aβ42 (10 μM), biflorin and derivatives (10 μM), curcumin (10 μM), and ThT (45 μM). 100 μl/well was placed in a 96-well black plate and fluorescence values were measured at 485 nm / 528 nm wavelength using a Synergy™ HTX multi-mode microplate reader, and all were performed three times.

3) Beta-amyloid western blot

All proteins were quantified using the BCA protein assay kit (Thermo). After quantification of Aβ42 protein, the protein structure was fixed through photo-cross linking. Proteins obtained by homogenization of 6-week-old mice brain samples were quantified and then electrophoresed by breaking disulfide bonds.

Proteins were separated by size at room temperature through SDS-PAGE, and transfer was performed with a PVDF membrane at 4°C for 2 hours. Blocking was performed with 5% bovine serum albumin (BSA) at room temperature for 1 hour and 30 minutes. The primary antibody was diluted 1:1000 in 2% BSA and allowed to stand overnight, and then reacted with the secondary antibody diluted 1:5000 in 1% BSA for 30 minutes at room temperature. The band was film detected using the ECL detection kit, and the signal was quantified with image J, an image analysis program. Actin was used as a loading control. Aβ antibody (sc-53822, Santa Cruz).

4) Beta-amyloid cytotoxicity test (MTT)

Neuro2a, a mouse neuroblastoma cell line, was purchased from the American Type Culture Collection (Menassas, VA, USA), and Minimum Essential Medium (WELGENE Co., Daegu, Korea) containing 10% fetal bovine serum and 1% penicillin streptomycin was used. . Cells were cultured in an incubator with air containing 5% CO2 at 37°C and 100% humidity, and subcultured when confluence reached 70-80%.

(1) Cell viability evaluation

Neuro2a cells were cultured in a 24-well culture plate (2×10 ^4/ well) for 24 hours, and then each drug concentration (0-30 μM) was treated for 24 hours. Aβ42 protein was pre-incubated for 24 hours to obtain aggregates and then treated with drugs. After drug treatment, the existing medium is removed, and the new medium is treated with MTT reagent (Thermo Fisher scientific) at a concentration of 0.5 mg/ml, reacted in an incubator at 37° C. for 30 minutes, then the medium is removed and the color development is induced with DMSO to induce a micro plate reader Absorbance was measured at 540 nm with (Bio-rad).

(2) Cytotoxicity evaluation

Neuro2a cells were cultured in a 24-well culture plate (2×10 ⁴ /well) for 24 hours, and then treated with Aβ42 aggregates and drugs at different concentrations (0-30 μM) for 24 hours. After drug treatment, the medium was collected and centrifuged at 1000 rpm for 2 minutes to recover the supernatant. The recovered supernatant was mixed 1:1 with LDH reagent in which solutions A and B were mixed at a ratio of 45:1, and reacted for 15 minutes in a light-shielding box, and then absorbance was measured at 540 nm using a microplate reader.

4) Statistical processing

All statistical analyzes and graphs were performed using Graphpad Prism 5.0. Neurite growth evaluation and passive avoidance test results were analyzed by one-way analysis of variance (ANOVA).

When significance was found by ANOVA analysis, each group was compared using post-hoc Turkey's pairwise comparison, and it was recognized as statistically significant in the range of p < 0.05.

2. Results

1) Inhibitory effect of beta-amyloid (Aβ) aggregation of Biflorin and derivatives

When only beta-amyloid was cultured, the aggregation inhibitory activity of biflorin derivatives was evaluated by setting the fluorescence intensity to 100. Biflorin (10 μM) showed about 19% inhibitory activity against aggregation of beta-amyloid (10 μM), biflorin #1 had no inhibitory activity at all, biflorin #2 82%, biflorin #3 91% , biflorin #4 exhibited an inhibitory activity of 37% and biflorin #5 of 8% (FIG. 1).

2) Effects of Biflorin Derivatives on Beta Amyloid Aggregate Decomposition

After culturing beta-amyloid for 24 hours to form aggregates, an experiment was conducted to determine whether the aggregates can be degraded by adding biflorin and derivatives. It was found that biflorin #2 and biflorin #3, which were excellent in inhibiting beta-amyloid aggregation, effectively decomposed the already formed beta-amyloid aggregates (FIG. 2).

3) Inhibition of beta-amyloid oligomer formation of Biflorin derivatives

It is known that beta-amyloid low-molecular oligomers (dimers and trimers) exhibit brain cytotoxicity, so the effect of biflorin and its derivatives on beta-amyloid low-molecular oligomer formation was evaluated. It can be seen that the production of dimer, trimer and tetramer is reduced when biflorin #2 and biflorin #3 are co-cultured compared to the case where only beta-amyloid is cultured. Curcumin, which was used as a positive control, also showed a similar decrease in beta-amyloid oligomers.

4) Cytotoxicity inhibitory effect of beta-amyloid of Biflorin derivatives

The effects of biflorin #2 and biflorin #3 that inhibit the production of beta-amyloid low-molecular oligomers on the cytotoxicity caused by beta-amyloid were investigated. It was found that both substances inhibit apoptosis by beta-amyloid (FIG. 4).

Claims (5)

A pharmaceutical composition for preventing or treating a disease caused by aggregation of beta-amyloid, comprising a compound of Formula 1 below:
[Formula 1]

(Wherein, R ₁ , R ₂ , and R ₃ are each independently hydrogen, a hydroxyl group, an alkoxy group having 1 to 3 carbon atoms, or glucopyranosyl; at least one of the three is not a hydrogen atom or a methoxy group ).
The composition of claim 1, wherein the compound of Formula 1 is at least one compound of Formulas 2 to 5 below:
[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

.
The method according to claim 1, wherein the compound of Formula 1 is a compound of Formula 3 or 4, the composition:
[Formula 3]

[Formula 4]

.

The composition of claim 1, wherein the disease is Alzheimer's dementia, Down's syndrome, amyloid angiopathy, systemic amyloid disease, Dutch type amyloidosis or inclusion body stomatitis.
A health functional food for preventing or improving diseases caused by aggregation of beta-amyloid in the brain, comprising the compound of Formula 1 below:
[Formula 1]

(Wherein, R ₁ , R ₂ , and R ₃ are each independently hydrogen, a hydroxyl group, an alkoxy group having 1 to 3 carbon atoms, or glucopyranosyl; at least one of the three is not a hydrogen atom or a methoxy group ).

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